Antifreeze proteins have been described in the literature, see for example Marilyn Griffith and K. Vanya Ewart in Biotechnology Advances, Vol 13, No 3, pp 375-402, 1995. Antifreeze proteins generally possess one or more of the following properties: thermal hysteresis, inhibition of ice recrystallisation, control of ice crystal shape and interaction with ice nucleators.
Thermal hysteresis is the best known property of AFPs and the property is normally used to test for the presence of AFPs. Thermal hysteresis results from a lowering of the apparent freezing temperature of a solution containing a thermal hysteresis active AFP without affecting the melting temperature. The identification of sources of AFP by thermal hysteresis tests is widely described in the literature, see for example John G. Duman in Cryobiology 30, 322-328 (1993).
Inhibition of ice recrystallisation is another property of AFPs. This activity is also referred to as ice crystal growth suppression. This property can be tested by comparing at a certain point in time the ice crystal size of crystals in the presence of AFP and in the absence of AFP. The application of this method in the testing of fish AFPs is described in U.S. Pat. No. 5,118,792 (DNA Plant Technology Corporation)
A third property of AFPs is their ability to influence the shape of ice crystals. This property stems from the selective binding of AFPs to certain faces of the ice crystal and therewith limiting crystal growth in certain directions. The presence of ice crystals having an hexagonal bipyramid shape is then considered indicative of the presence of AFP. This method is for example described for testing the activity of extracellular winter rye AFPs in WO 92/22581 (University of Waterloo).
A fourth property of AFPs is their ability to inhibit the activity of ice nucleating substances. This interaction between an AFP and an ice nucleator may for example result in increased thermal hysteresis. This property is for example tested in WO 96/40973 (University of Notre dame du Lac)
AFPs have been suggested for improving the freezing tolerance of products. Many applications have been suggested in this context.
For example AFPs have been suggested for enhancing the cryopreservation of biological materials (WO 91/12718, Agouron Pharmaceuticals, WO 91/10361, The Regents of the University of California). Also AFPs have been suggested to prevent leakage from liposomes e.g. in cosmetic or pharmaceuticals (see WO 96/20695). A further possible application is to increase the freezing tolerance of plants by including therein (or transgenetically producing therein) an AFP (See J. Cell. Biochem. Suppl. vol. 14e, 1990, page 303 XP002030248, Lee et al, abstract R228). Also fish AFPs have been suggested for use in food products for example in frozen yoghurt or ice cream (U.S. Pat. No. 5,620,732 Pillsbury and WO 96/11586, HSC Research and development limited partnership).
Up till now, however the use of AFPs has not been applied on a commercial scale. Applicants are of the opinion that one of the reasons for the lack of commercial implementation is that although many AFPs have been described, in practice the implementation in actual commercial products encounters serious problems.
Applicants have found that one of the key reasons for these problems is that out of the great number of AFPs that have been described in the literature only a limited set of AFPs can suitably be applied for each application; also applicants have found that this selection of suitable AFPs is dependent on the desired application and/or product attributes to be achieved.
A particular desirable source of AFPs is plant material. Plant materials can fairly easily be obtained in relatively large quantities and relatively simple isolation procedures can be used for obtaining an AFP containing concentrate. Furthermore the use of AFPs from plant material is believed to be favoured by consumers who tend to prefer natural vegetable sources to e.g. fish AFPs.
Marilyn Griffith and K. Vanya Ewart in Biotechnology Advances, Vol 13, No 3, pp 375-402, 1995 have given a list of 27 higher plant species in which antifreeze activity is found. This article also suggests a wide range of possible applications for AFPs.
Applicants have now found that if a specific application for the use of plant AFPs is selected, this creates the need for a specific test to select the limited set of AFPs which can advantageously be applied in this application.
The object of the present invention is therefore to provide those plant AFPs which can advantageously be used in frozen confectionery products.
Surprisingly, applicants have found that despite the fact that a great number of plants contain AFPs, only a limited set of plants contains AFPs which are capable of providing a good texture to frozen confectionery products. Surprisingly it has been found that a relatively simple test method can be used to select the suitable AFPs.
Accordingly in a first aspect the invention relates to frozen confectionery products comprising one or more AFPs derived from plants, wherein the AFPs in water have an ice crystal size after quick freezing to -40.degree. C. followed by storage for 1 hour at -6.degree. C. (measured as described below) of less than 15 .mu.m.